This invention relates to enzyme compositions comprising whole microorganism cells, to methods of making such compositions, and to methods for using such compositions to carry out enzymatic reactions. More particularly, this invention relates to an improved glucose isomerase enzyme composition comprising whole microbial cells, to methods of making such compositions, and to methods of using such compositions for isomerizing glucose to fructose.
U.S. Pat. Nos. 3,821,086 to Lee et. al., and 3,935,069 to Long, disclose processes for preparing flocculated whole enzyme-containing microorganism cells and for using such cells in continuous column operations to carry out enzymatic reactions. Preparation of whole flocculated Arthrobacter cells containing glucose isomerase and the use of such cells to isomerize glucose to fructose are among the processes disclosed. U.S. Pat. No. 4,060,456 to Long also discloses the preparation of whole flocculated Arthrobacter cells containing glucose isomerase and the use of such cells to isomerize glucose to fructose.
The use of whole flocculated enzyme-containing microorganism cells for carrying out enzymatic reactions represents a major improvement over the use of cell-free enzyme extracts. Flocculated whole cells can be used in continuous column reactions and can also be used repeatedly in batch reactions. Cell free extracts, on the other hand, are generally water soluble and therefore can be used only once and only in batch reactions. The use of flocculated whole cells also represents a significant improvement over the use of non-flocculated whole cells containing enzymes, as disclosed for example in U.S. Pat. No. 3,817,832 to Lloyd et. al., because flocculated cells can be used in continuous columns while non-flocculated cells are suitable for use only in shallow beds.
Although the use of flocculated whole microorganism cells represents an important step forward in enzyme technology, such cells are subject to compaction under high hydrostatic heads, so that the column height must be limited. U.S. Pat. No. 3,935,069 to Long, cited supra, describes the preparation and use of hardened flocculated microbial cell aggregates, in which magnesium oxide or other inorganic compound (i.e., an oxide, hydroxide, phosphate or sulfate of magnesium, calcium, iron or manganese) is added to a fermentation broth or cell susspension just prior to the addition of flocculants. Comparative test data show that the flow rate of glucose solution through magnesium oxide-hardened Arthrobacter cells occurs much more slowly than through non-hardened Arthrobacter cells under similar test conditions. The preparation and use of enzyme compositions comprising an enzyme attached to a support or carrier is also well known. Numerous references describe immobilized enzymes of this type. Carriers may be hard material such as porous glass or polystyrene beads, or materials affording a high void volume such as regenerated cellulouse (see U.S. Pat. No. 4,029,546 to Brouillard); immobilized enzymes on either type of carrier are suitable for continuous column operations. However, preparation of enzymes immobilized on a carrier requires extraction and purification of the enzyme to obtain a cell-free extract, followed by binding the enzyme to the carrier, usually with the aid of a binding agent. Whole cell enzyme compositions are more desirable, since they do not require these processing steps, provided the whole cell compositions are sufficiently hard for use in a column of substantial height.